Single-molecule investigations of fast biomolecular search processes initiating DNA repair

我的研究计划的首要目标是建立对快速分子搜索过程的新的机械论理解，这是一系列关键细胞过程的基础。一类快速的分子搜索过程由于分析技术的限制在很大程度上仍未确定，即在DNA被电离辐射、自由基和复制过程切割后启动持续的DNA修复过程的搜索。现有的显微镜技术不能在生理条件下描述这些分子搜索的特征，因为它们不能同时解决：弱的蛋白质-DNA和DNA-DNA相互作用；单个分子在几秒钟内的扩展搜索轨迹，以及毫秒-时间尺度的相互作用动力学；它们也不能与拓扑复杂或应变的DNA底物兼容。我将使用一套新的定制单显微镜技术来扩展CLIC成像，从而绕过这些限制。 我的第一个短期目标是创建一个独特的体外显微镜站，能够解决有关校准良好的MRX修复蛋白复合体执行的分子搜索的公开问题，其生物物理性质和DNA相互作用以前已经被测量过，尽管在有限的条件下。我的第二个短期目标是应用这些技术对9-1-1修复蛋白复合体进行第一次单分子研究，该复合体只被用生化、细胞生物学和遗传分析进行研究。我的长期目标是扩展CLIC成像，创建一个独特的力和荧光体外显微镜站，我将应用它来探测DNA底物上受控制的拓扑和作用力的分子搜索。这项研究计划为在系统接近复杂细胞环境的条件下对广泛的分子搜索过程(例如信使RNA的转录和翻译)进行首次单分子研究奠定了基础，并在生物医学研究中有着广泛的应用。

The overarching objective of my research program is to create a new mechanistic understanding of the fast molecular search processes, which underlie a host of key cellular processes. One class of fast molecular search processes, that remain largely uncharacterized due to limited analysis techniques, is the search that initiates the constant processes of DNA repair following its cleavage by ionizing radiation, free radicals and during replication. Existing microscopy techniques are not capable of characterizing these molecular searches under physiological conditions because they can not simultaneously resolve: weak protein-DNA and DNA-DNA interactions; extended search trajectories of individual molecules over several seconds, and millisecond-timescale interaction kinetics; nor are they compatible with topologically complex or strained DNA substrates. I will circumvent these limitations using a new suite of custom single-microscopy techniques which extend CLIC-imaging. My first short-term objective is to create a unique in vitro microscopy station, capable of addressing open questions about the molecular searches performed by the well-calibrated MRX repair protein complex, whose biophysical properties and DNA-interactions have been measured previously, though under a limited range of conditions. My second short-term objective is to apply these techniques to perform the first single-molecule investigations of the 9-1-1 repair protein complex, which has only been studied with biochemical, cell-biological and genetic assays. My long-term objective is to extend CLIC-imaging to create a unique force and fluorescence in vitro microscopy station, which I will apply to probe molecular searches on DNA substrates of controlled topology and applied force. This research program sets the stage for the first single-molecule investigations of a wide range of molecular search processes (e.g. transcription and translation of messenger RNA) under conditions that systematically approach the complex cellular milieu, with myriad applications to biomedical research.